1. Field of the Invention
The subject invention pertains to a unique method of manufacturing a mixture of amplified double-stranded nucleic acids comprising unknown sequence.
2. Discussion of the Background
The 5′ End of Nucleic Acid Strand and the Methods to Capture it
The precise determination of the sequence around each 5′ end of nucleic acids is of a special interest in biology. In the case of ribonucleic acid (RNA), this sequence reveals a corresponding location in the chromosomes, the gene promoter, which is a crucial component of the gene regulation mechanism. Therefore, many efforts have been made to isolate 5′ ends of RNA molecules. However, it is difficult to be certain that the observed 5′ end corresponds to a molecule really existing in vivo and is not the product of the truncation of a longer RNA molecule during the experiment.
Several approaches have taken advantage of the fact that the 5′ ends of the complex fraction of the total RNA content of living cells—comprising the messenger RNAs (mRNAs)—can be distinguished by the presence of a specific modification, called the cap, which consists of a guanosine 5′ triphosphate modified by one or more methylations. In experimental conditions, RNA molecules bearing the cap can be considered intact in their 5′ end, and methods aimed to capture the 5′ ends ensure that only these molecules participate to the final product. For instance, in the Cap-Trapper method, the diol group of the cap is used to bind 5′-intact RNA molecules on beads and to exclude complementary deoxyribonucleic acids (cDNAs) of truncated RNA molecules [Caminci 2001 the disclosure of which is herein incorporated by reference in its entirety.]. In the oligo-capping method, a 5′ oligonucleotide is attached to the RNA molecules in three reactions using phosphatase, pyrophosphatase and ligase [Maruyama 1994, the disclosure of which is herein incorporated by reference in its entirety.]. In the CAPswitch method, the propensity of the reverse transcriptase (RTase) to add extra cytidine nucleotides to the first-strand cDNAs templated from capped molecules is exploited to distinguish them from the uncapped RNAs [Chenchik 1999, U.S. Pat. No. 5,962,272, the disclosure of which is herein incorporated by reference in its entirety.].
The CAPswitch Method
Chenchick et al invented the CAPswitch method, “for the synthesis and cloning of full-length cDNA, or cDNA fragments, that correspond to the complete sequence of 5′-ends of mRNA molecules”. Pivotal in this method is the use of a template switching (TS) oligonucleotide that serves as a new template to extend the first-strand cDNAs after they reach the cap of their mRNA template. The mechanism of CAPswitch was not clearly understood at the when this method was patented. The optimal sequence at the 3′ end of the TS oligonucleotide was determined by Chenchick et al by using TS oligonucleotide finishing with random nucleotides and analyzing the sequence of the resulting full-length cDNAs. It was then refined by a mutagenesis analysis. They hypothesized that the TS oligonucleotide would hybridize to the cap structure of the mRNA (see the CAPswitch patent's abstract and figures), or to extra 3′ cytidine residues on the first-strand cDNA added by a terminal transferase activity of the RTase. The role of the CAP was unclear, as it was stated that it was not a necessary requirement for strand template-switching, but was making the reaction more effective for full-length
In fact, It has been demonstrated that the mechanism for first-strand cDNA extension depends on the molecular nature of the cap, because it is used by the RTase as a template for a short extension of the full-length cDNA: cytosines are added for 7-methylguanosine caps [Hirzmann 1993, the disclosure of which is herein incorporated by reference in its entirety.], but in the case of adenosine caps (very frequent intermediate steps in enzymatic reactions), the extra nucleotides added are thymidines [Ohtake 2004, the disclosure of which is herein incorporated by reference in its entirety.]. A residual terminal transferase activity is detected for cap-less RNA molecules and as a 5% background for capped molecules. This explains why only cDNAs that reached the 5′ end of a RNA molecule can switch template.
The patent of Chenchick et al is written broadly and covers usage of total RNA or polyA-tailed RNA, the use of oligodT or random RT primers, the specific amplification of one gene or the amplification of a whole library.
However, eight years after it was issued, there is no academic or commercial evidence that the patented method can be used to amplify a library of complete 5′ ends of capped RNA (with and without polyA tails) using random RT primers on total RNA at nanogram scale. In the example number 1 of the patent, a library is made using random primers, but together with polyadenylated RNA. In example 2, a library is made from 100 nanograms of total RNA, but using oligodT RT primers. In example number 3 polyadenylated RNA is reverse-transcribed with a double stranded oligodT primer. In example number 4, total RNA is reverse-transcribed by oligodT primers, and the subsequent steps include a enzymatic cleavage in which the 5′ completeness is lost. The patent does contain an example of usage of CAPswitch with total RNA and random RT primers. However, it is in the context of the “5′ rapid amplification of cDNA ends” method, which is gene-specific: the products of the RT are not amplified as a library.
Consequently, simultaneous practical usage of random RT primers and total RNA for preparing a library of complete 5′ ends is absent in the patent or in the later literature, and thus the RT products having the complete 5′ ends are not amplified as a library.
Suppressive PCR
Suppressive PCR is known as a method used for the preparation of libraries of target nucleic acids in a complex nucleic acid mixture and used to selectively control the size of PCR products which can be amplified in a reaction.
Suppressive PCR was invented by Chenchik et al, who were issued the U.S. Pat. No. 5,565,340 in 1996 ([Chenchik 1996, the disclosure of which is herein incorporated by reference in its entirety.]). In this method, some DNA molecules are prevented from being amplified during a PCR by adding complementary adapters to their 5′ and 3′ ends. The complementarity between the adapters exceeds the complementarity between adapters and PCR primers. During the annealing step of the reaction, intramolecular folding will be favored compared to hybridization with PCR primers, and the folded templates will not be extended. Due to the exponential nature of the PCR, when a significant number of molecules skip amplification at every cycle, their contribution to the final product becomes neglectable.
The strategies of suppressive PCR can be classified in two categories. In the first, the PCR is made with a pair of different forward and reverse primers, and each adapter have complementarity to one of them. Therefore, the templates flanked by the same adapter will be suppressed, whereas templates that match both PCR primers will be amplified.
In the second category, the PCR is made with a single primer. In this case, the molecules that will be amplified are the ones for which the intramolecular folding is the less probable: the longer molecules. This approach is usually employed to suppress primer dimers and counterbalance the tendency of the PCR to favour the shortest amplicons [Brownie 1997, the disclosure of which is herein incorporated by reference in its entirety.].
To summarize, there are two possible strategies of suppressive PCR with distinct advantages: the first strategy described above results in a sequence-based selection while the latter strategy gives rise to a size-based selection, trying to overcome the bias towards short-sized templates that commonly affects PCR.
However, it is unfortunately difficult to get all the benefits of the suppressive PCR method at the same time: to normalize the size of the amplicons and eliminate primer dimers, the templates should have the suppressive sequences in 5′ and 3′, but when the method is used to eliminate other artifacts, the desired templates should be amplified with distinct forward and reverse PCR primers.
The reason why it was very difficult to combine total RNA, CAPswitch and random priming for the preparation of libraries of complete 5′ ends from nanograms of template is the artifacts created during the RT. Since only low quantities of template are used, the second-strand cDNA synthesis should be coupled with an amplification step, typically PCR, in which the artifacts will outcompete the 5′-complete cDNAs. Artifacts can be created in the following situations:                TS oligonucleotides can compete with RT primers for binding the RNA molecules, especially at the cool temperatures used for annealing the random RT primers. (See FIG. 48A)        RT primers can invade the DNA-RNA duplex when the RTase pauses (this enzyme is not very processive), and terminate the reaction by premature template-switching. (See FIG. 48B)        As 25% of the random RT primers finish by a guanine, they can compete with the TS oligonucleotides for binding the extra cytosines added in 5′ of the first strand cDNA by the RT. (See FIG. 48C)        TS oligonucleotides can hybridise with the random RT primers, and the RTase can extend these complexes. (See FIG. 48D)        Random RT primers hybridize each other can and be extended as well. (See FIG. 48D)        
At first the inventors thought to use suppressive PCR to avoid the amplification of the primers dimers, by giving them a sequence tail designed according to Chenchick et al's method and using single universal PCR primer.